Genome-wide identification of starch phosphorylase gene family in Rosa chinensis and expression in response to abiotic stress

Chinese rose (Rosa chinensis) is an important ornamental plant, with economic, cultural, and symbolic significance. During the application of outdoor greening, adverse environments such as high temperature and drought are often encountered, which affect its application scope and ornamental quality. The starch phosphorylase (Pho) gene family participate in the synthesis and decomposition of starch, not only related to plant energy metabolism, but also plays an important role in plant stress resistance. The role of Pho in combating salinity and high temperature stress in R. chinensis remains unknown. In this work, 4 Phos from R. chinensis were detected with Pfam number of Pho (PF00343.23) and predicted by homolog-based prediction (HBP). The Phos are characterized by sequence lengths of 821 to 997 bp, and the proteins are predicted to subcellularly located in the plastid and cytoplasm. The regulatory regions of the Phos contain abundant stress and phytohormone-responsive cis-acting elements. Based on transcriptome analysis, the Phos were found to respond to abiotic stress factors such as drought, salinity, high temperature, and plant phytohormone of jasmonic acid and salicylic acid. The response of Phos to abiotic stress factors such as salinity and high temperature was confirmed by qRT-PCR analysis. To evaluate the genetic characteristics of Phos, a total of 69 Phos from 17 species were analyzed and then classified into 3 groups in phylogenetic tree. The collinearity analysis of Phos in R. chinensis and other species was conducted for the first time. This work provides a view of evolution for the Pho gene family and indicates that Phos play an important role in abiotic stress response of R. chinensis.

have significant sequence similarities with each other, but they differ in molecular size, substrate specificity, physiological effects, and intracellular localization 7 .Starch phosphorylase has been widely studied in Arabidopsis thaliana 8,9 , Oryza sativa 10 , Zea mays 11 , wheat (Triticum aestivum L.) 12 , and potato (Solanum tuberosum L.) 13 .In A. thaliana, AtPho1 seems to play a role in abiotic stress rather than in starch degradation 8 .The role of Pho in starch synthesis and decomposition has been widely researched, while the gene family's action in stress resistance via carbohydrate metabolism is worth further research 8,14 .
Roses (Rosa chinensis) is an important ornamental plant, with economic, cultural, and symbolic significance 15 .They are used not only as fresh cut flowers but also widely used as outdoor landscaping flowers 16 .During the application of outdoor greening, adverse environments such as high temperature and drought are often encountered, which affect its application scope and ornamental quality 17 .In the study of drought stress in roses, it was found out that with the deepening of drought, the content of proline and soluble sugar increased 18 .Phytohormone signal transduction was also a pathway in regulating the salt tolerance response of Rosa chinensis 19 .Phos play an important role in the synthesis and decomposition of starch, not only related to plant energy metabolism, but also to plant stress resistance 8,14 .The role of Phos in combating high temperature and drought treatment in R. chinensis is unclear.Thus, we identified the Phos family in R. chinensis and analyzed the expression levels of these genes under different stress treatments.This work provides new insights on the Pho gene family response to abiotic stress.

Identification of Pho gene family
Fourteen genomes were obtained from public genetic data services (Table 1), for detailed information, see Supplementary Table S1.The HMMER (v3.2) 20 and Pfam number of Pho (PF00343.23)were used to search Pho protein sequence (E-value < 1e −10 ).In order to minimize the deviation caused by different gene annotation methods, we used homolog-based prediction (HBP) as a supplement tool to predict genes (Supplementary Table S2) 21 .Genes with short annotation were merged along with adjustments of the promoter region.The resulted genes were tested for the functional annotation by KEGG (https:// www.genome.jp/ kegg/) and Swissprot (release-2021_03) (http:// www.ebi.ac.uk/ sprot).The motif was recognized using Meme (https:// meme-suite.org/ meme/ tools/ meme), with a number of 11 22 .The domain was predicted using CDD (https:// www.ncbi.nlm.nih.gov/ cdd/) database.Enzyme function prediction was performed using CLEAN software (https:// clean.platf orm.molec ulema ker.org/ confi gurat ion) 23 .

Sequence alignment and phylogenetic analysis
Protein sequence alignments were conducted by muscle 5.1 39 .The phylogenetic tree was then constructed using iqtree (v1.5.5) under the MFP model 40 , using maximum likelihood with parameters of 1000 replicates of bootstrap, the results were displayed by the Interactive Tree of Life (itol), an online tool for the display, annotation and management of phylogenetic trees (https:// itol.embl.de/ upload.cgi).

Physicochemical properties and protein structure prediction of Pho in R. chinensis
Database of ExPASy (http:// expasy.org/ protp aram/) 41 was used to predict the number of amino acids, sequence coding for amino acids in protein (CDS), isoelectric point (Acidity < 7, alkalinity > 7), molecular weight,

qRT-PCR Analysis of Phos response to high temperature and salt
Two-year-old cutting seedlings of R. chinensis used in the experiments were purchased from Huaihua flower market (109.938318°E, 27.410230° N) and identified by Prof. Liang Cao.The plant materials, nearly identical in size, were planted in the pots (20 cm in diameter, 35 cm in height) filled with mixed sterilized soil of nutritive soil, garden soil, perlite, and vermiculite in the ratio of 5:3:1:1 (v/v).Before high temperature and salt treatment, all samples were cultured under conditions of 70% relative humidity and day/night temperatures of 25 °C/20 °C in the same artificial climate chamber at Hunan University of Medicine.Subsequently, the samples were subjected to a high temperature (45 °C) and salt (0.4%, NaCl: Na 2 SO 4 = 1:2 in molar) treatment for 2 h.Each treatment group consisted of three biological replicates.Total RNA was extracted from the fourth or fifth fully expanded leaves in control (CK), high temperature and slat treatment, using an OminiPlant RNA Kit (CWBIO., Jiangsu, China) and a PrimeScript™ RT reagent Kit with gDNA Eraser (TransGen Biotech, Beijing, China) for reverse transcription synthesis of complementary DNA, following the manufacturer's instructions.Four Phos from R. chinensis were selected for qRT-PCR analysis.GAPDH was used as the reference gene, and the primer sequences are presented in Supplementary Table S4.The fluorescence signals were detected using a LightCycle 480 Real-Time System (Roche Diagnostics Ltd., Rotkreuz, Switzerland) after configuring the PCR reaction had with PerfectStart Green qPCR SuperMix (TransGen Biotech, Beijing, China).For each biological replicate, three technical replicates were performed, respectively.

Data analysis
The primary outcomes will be compared between the groups using a t-Test in SPSS (Statistical Package for the Social Sciences) software, and an analysis of variance for repeated measurements will be used for data analysis.

Plant guideline statement
Experimental research and field studies on plants (either cultivated or wild), including the collection of plant material, comply with relevant institutional, national, and international guidelines and legislation.

Identification of Pho gene family
Sixty-three Phos were retrieved from fourteen genomes, namely V. Vinifera, M. truncatula, Z. Jujuba, C. sativa and ten species from Rosaceae family including R. chinensis.Among them, the short annotations of MP16G1059300 and MP16G1059400 merged into MP16G1059300m, Cpi13g547 and Cpi13g548 merged into Cpi13g547m by using HBP analysis.1), in total, 69 Phos from 17 species were classified into three groups, which fit with the functional annotation of SP1 (alpha-1,4 glucan phosphorylase), SP2 (alpha-glucan phosphorylase) and SP3 (glycogen phosphorylase 1-like).The group of SP1 contains three subgroup, namely clades A1 to A3.In clade of A1, V. vinifera, A. thaliana, and nine species of rosids were single copy, while C. pinnatifida, P. Pyrifolia, Malus × domestica and M. prunifolia were double copies.In A2 clade, twelve species with single copy were gathered.Single copy form O. sativa and Z. mays were classified in clade A3.SP2 contains two clades of B1 and B2, in clade B1, V. vinifera, A. thaliana, and nine species of rosids were single copy, while for species of C. pinnatifida, P. pyrifolia, Malus × domestica, two copies for each of them, and three copies for M. prunifolia.In clade B2 contains single copy for each of O. sativa and Z. mays.In the SP3 clade, V. vinifera and thirteen other species were single copy.

Physicochemical properties of Pho in R. chinensis
Four copies of Phos were screened from R. chinensis in this work, Rco2g0166331, Rco4g0438671, Rco5g0009141, and Rco5g0068321, with lengths ranging from 821 to 997 base pairs and 14 to 22 CDSs.The calculated theoretical pI < 7, aliphatic index < 100, and GRAVY < 0, indicate weak acidity and hydrophilicity.In the subcellular localization analysis, Rco2g0166331 was deduced to be located in the mitochondrion, Rco4g0438671 in the chloroplast, and Rco5g0009141 and Rco5g0068321 were assumed to be expressed in the cytoplasm (Supplementary Table S5).
The calculation of dN/dS (ω < 1) indicates negative selection, and eleven common motifs of these four Phos were identified (Fig. 2A), and the GT35_Glycogen_Phosphorylase domain was contained in the proteins, showing high consistency in sequences (Fig. 2A; Supplementary Table S6).

Promoter cis-acting elements and protein structure analysis of Phos in R. chinensis
In order to investigate the biological functions of the Pho gene family in the growth, development, and stress resistance of R. chinensis, we analyzed the 2.0 kb sequence upstream of the Phos and identified cis-acting elements consistent with the gene transcription direction (Fig. 2B).All predicted cis-acting elements were classified into   also detected.Five cis-acting elements: A-box, CAT-box, CCG TCC motif, CCG TCC -box, and O 2 -site, which participate in plant growth and development were screened.Among these, the CAT box and CCG TCC motif are cis-acting elements related to meristem-specific activation.
The functional domains of Pho were aligned, then the protein structure of Phos were predicted (Fig. 2C,D), revealing that SP1 differrs from SP2 and SP3 by an L80 insertion.There is also a significant difference in the L80 insertion domain between monocots and dicots, as well as a significant variation between R. chinensis and A. thaliana (Fig. 2C).The PLP is phosphorylase pyridoxal-phosphate attachment site, all known phosphorylases share the catalytic and structural properties, and the sequence is highly conserved.We discovered that Rco2g0166331and Rco4g0438671 shared the same PLP sequence with OsPho1 and MzPho1, while Rco5g0068321 and Rco5g0009141 have variations of 2 and 4 amino acids, respectively (Fig. 2C).The variation in genetic sequences resulted in different three-dimensional (3D) protein structures, with two copies of SP1(Rco2g0166331, Rco4g0438671) showing similar structures, distinct from those of SP2 (Rco5g0068321) and SP3 (Rco5g0009141) (Fig. 2D).

Average nucleotide identity and intergenomic collinearity of Pho gene family
Average nucleotide identity (ANI) between R. chinensis and eleven other genomes were summarized (Fig. 3A).R. chinensis have close ANI of 0.93 to R. rugosa, as they are from the same genus.The ANI value in 0.75-0.83between R. chinensis and other species.P. pyrifolia and C. pinnatifida, M. prunifolia and C. pinnatifida, M. prunifolia and P. pyrifolia have ANI value of 0.90, 0.90, 0.91 respectively, means they are close in genetic background.Collinearity analysis of Phos in R. chinensis, R. rugosa, R. chingii, and F. vesca were conducted using jcvi (v1.1.12) 45software (Fig. 3B).The block where the Phos are located exhibits good collinearity among four species, and there is chromosomal segment inversion between R. chinensis and R. rugosa.Tbtools was used to investigate the collinearity relationship of Phos among the genomes of R. chinensis, V. vinifera, and Z. jujuba (Fig. 3C).Rco2g0166331 and Rco4g0438671 each have two homologous copies in the genome of V. vinifera, while Rco2g0166331 have no homologous copy in Z. Jujuba.We analyzed the homology of 6 linkages genes both upstream and downstream of the Pho gene family (Fig. 3D).Rco2g0166331 and Rco4g0438671 have three homologous genes upstream, Rco5g0068321 have one homologous gene downstream, while Rco5g0009141 have no homologous gene.To elucidate the expression patterns of the Pho gene family of R. chinensis under stress treatment, we analyzed its gene expression levels under conditions of drought, salinity, high temperature, and different phytohormone treatments (Fig. 4A).Rco5g0009141 was significantly upregulated after 30 and 60 days of drought treatment, while Rco4g0438671 was upregulated after 30 days of drought treatment (Fig. 4B).Rco2g0166331, Rco4g0438671, and Rco5g0068321 were significantly downregulated after 90 days of drought treatment.After 2 h of salt treatment, the expression levels of Rco2g0166331 and Rco5g0068321 were significantly upregulated, whereas the expression levels of Rco5g0068321 were significantly downregulated after 48 h of treatment.Under high temperature treatment (45 °C), Rco5g0068321 was significantly downregulated at 2 h, 6 h, and 12 h, while Rco2g0166331, Rco4g0438671, and Rco5g0009141 were significantly downregulated under 6 h of treatment.The expression levels of Phos under different phytohormones treatment for 24 h were analyzed.It was demonstrated that there was no significant change in four Phos under 100 μM abscisic acid (ABA) treatment.The expression levels of Rco2g0166331, Rco5g0009141, and Rco5g0068321 were significantly up-regulated after 50 μM jasmonic acid (JA) treatment, and all four Phos were significantly up-regulated under 100 μM salicylic acid (SA) treatment.
To conform the transcription level of the Phos, high temperature and salt treatment were administered for two hours each.The four Phos were amplified by using qRT-PCR (Fig. 4C).Under salt treatment, Rco2g0166331 and Rco5g0068321 were significantly upregulated, consistent with the transcriptome data results.While four Phos were significantly downregulated under 2 h of high temperature treatment, only one gene (Rco5g0068321) was significantly downregulated in transcriptome data (the other 3 genes were also downregulated), indicating that all Phos respond to high temperature stress.

Discussion
During the process of evolution, it is common for plants to undergo genome doubling or gene amplification events to adapt to environmental changes.The generated duplicate genes are an important source of genome evolution, and provide the foundation for regulation and function diversity 54,55 .Grape has not undergone polyploidization since the γ event 56 .Similarly, jujube has also not undergone WGD after the γ event 57 .On the other hand, pears and apples have undergone recent whole-genome duplication events 58 .All these species belong to the rosids.Based on the results of the phylogenetic tree (Fig. 1), we believe that the three groups SP1, SP2, and SP3 are the result of whole-genome triplication in rosids.
According to the phylogenetic tree, Rco4g0438671 (SP1) and Rco5g0068321 (SP2) are believed to be essential for maintaining metabolism.Rco2g0166331 (SP1) may be redundant copy produced by the expansion or replication of Rco4g0438671, based on the collinearity (Fig. 3C) and gene linkage (Fig. 3D), with the expression levels of the two genes tending to be consistent under different treatments (Fig. 4A).Rco5g0009141 (SP3) may be a copy derived from genome duplication 59 .Therefore, the upstream and downstream regions of Rco5g0009141 do not share syntonic blocks with other genes, and sequence variation also observed due to long-term evolution.The expression level of Rco5g0009141 was lower than that of Rco5g0068321 under different stress treatments (Fig. 4A), and the latter is necessary for maintaining cellular metabolism.Therefore, Rco5g0009141 can undergo relatively greater arbitrariness evolution.
Carbohydrates play an important role in maintaining plant's physiology conditions and coping with abiotic stresses.Rco4g0438671 and Rco5g0009141 were significantly upregulated after 30 days of drought treatment, accelerating starch decomposition to improve drought resistance.After 90 days of drought treatment, the leaves severely curled and withered, Rco2g0166331, Rco4g0438671, and Rco5g0068321 were significantly downregulated 3 .The expression levels of Phos were upregulated after 2 h of salt treatment, particularly Rco2g0166331 and Rco5g0068321, which were significantly upregulated, participating in starch catabolism to maintain cellular osmotic balance.Under high temperature stress, the expression levels of four Phos were significantly downregulated at 6 h of treatment, and in qRT-PCR analysis, the transcription level were observed to be significantly downregulated as early as 2 h into the treatment, indicating the Phos response in high temperature in maintain the carbon balance and energy homeostasis 60 .Under treatment with 50 μM JA and 100 μM SA, the expression levels of four Phos were upregulated, indicating that these genes can respond to phytohormone signals e involved in plant growth and stress resistance.The cis-acting elements of as-1 and TCA-element, responsive to SA were detected in the gene structure, indicating the involvement Phos in the hormone-mediated stress resistance.Many light-responsive cis-elements also detected in the Phos, suggesting that the expression of these genes may be related to photosynthesis progression 17 .The Phos can respond to abiotic stress factors providing new insights into the stress resistance mechanisms of R. chinensis.

Conclusions
The starch phosphorylase (Pho) genes are involved in the reversible process of starch synthesis and decomposition.In this work, 69 Phos from 17 species were gathered to construct the phylogenetic tree, the genes were classified by catalyze activity.The structure of Phos in R. chinensis were determined by using bioinformatic method.The expression levels of Phos under different stress treatment were analyzed using transcription database and qRT-PCR experiment.The results shown that Phos can respond to abiotic stress factors such as drought and salinity, as well as phytohormone signals.This work provides new insights into the role of Phos in stress resistance in R. chinensis.

4
types: abiotic and biotic stress, light response, phytohormone response, and plant growth and development (Fig.2B).MYB binding site (MBS) (drought-inducibility) were detected in Rco2g0166331 and Rco4g0438671.The stress-responsive element (STRE, AGGGG) for heat and osmotic stress were detected in Rco5g0009141 and Rco5g0068321.The low/hypoxia stress element GC motif and low temperature response element TCA were only present in Rco2g0166331.TC rich repeats for defense were only found in Rco5g0009141, and MYB and MYB-like sequences were detected in all four Phos.ACE, ARE, Box 4, G-Box and GT1-motif and other light response elements were widely distributed among the four Phos.In the phytohormone-responsive cis-acting elements, we detected abscisic acid-responsive elements (ABRE) responsive to abscisic acid, as-1 and TCA-element responsive to salicylic acid, and CCAAT-box responsive to ethylene synthesis.CGTCA-motif and TGACGmotif involved in the MeJA-responsiveness, estrogen response element (ERE) involved in the estrogen-response, TATC-box involved in the gibberellin-responsiveness, and TGA-element involved in auxin responsiveness were

Figure 4 .
Figure 4. Expression patterns of the Phos in different stress treatment.(A) Heat map of 4 Phos under different stress treatment.(B) Statistic of expression level of 4 Phos under different stress treatment (GCK: control of drought treatment, GH30, GH60, GH90: drought treatment of 30 days, 60 days, and 90 days, respectively; YCK: control of salt treatment, YT02, YT24, YT48: salt treatment for 2 h, 24 h, and 48 h, respectively; HCK: control of high temperature treatment, Hot02, Hot06, Hot12: high temperature treatment for 2 h, 6 h and 12 h respectively; PCK: control of phytohormone treatment, ABA, JA, and SA represents abscisic acid, jasmonic acid, and salicylic acid treatment for 24 h respectively).(C) Transcription of Phos in hot and salt treatment.

Table 1 .
The genomes information of 17 species and 69 Phos identified.